This proposal is the logical continuation of an exploratory collaborative grant (R21,Herold - von Herrath) focused to develop novel combinatorial approaches of recent-onset type 1 diabetes (T1D). Forthcoming results in two independent diabetes models (NOD and RIP-LCMV) have established the concept that, in order to achieve antigen-specific tolerance, combination of immunization with islet antigens and systemically acting immune modulators can exhibit strong synergy and be clinically beneficial for the following reasons: First, reversion of hyperglycemia can occur at lower dosages of the systemically acting immune modulator, in our case anti-CD3 Fab'2. Second, mechanistically, the induction of Tregs specific for islet antigens that can mediate long-term tolerance and bystander suppression is enhanced. This project seeks to deepen our mechanistic insight, and, in close collaboration with projects 2 and 3, address crucial issues that should facilitate translation of combinatorial therapy in recent-onset T1D to the clinic. Wewill answer the following questions: 1. Which is the optimal combinatorial therapeutic regimen in recent-onset T1D in vivo? Current data indicate that oral or nasal administration of insulin peptides bears most promise. In order to optimally tie into current choices in drug development, we will define the best candidate. In addition we will explore combination of antigen-specific therapy with GLP-1 agonists and gastrin to regenerate beta cells. 2. Which precise functions define the action of the islet antigen induced Tregs in vivo? Current findings show that long-term tolerance after anti-CD3 and antigen administration is, to a large part, due to induction of potent islet antigen-specific regulatory T cells (Tregs) that can transfer tolerance to recipients with recent-onset T1D. Their precise mechanism of action will be defined using novel technology and reagents recently acquired, RNAi and ins-TcR transgenic mice. 3. Which are optimal in vitro assays to monitor Tregs and antigen-sepcific tolerance in vivo? We will establish asays that reflect and predict the clinical out come on a per-animal basis. These assays should provide strong guidance to the goals of the clinical project (#3, Herold).